Several biomedical assays use fluorescent probes or fluorescent labeling due to the specificity and sensitivity that these techniques provide for detection, sensing, and imaging tasks. A major obstacle in using fluorescent labeling for cytometric analysis of cells in bodily fluids is the need for special sample preparation steps, since most of these fluids are optically dense and light scattering, thus it is problematic to excite the fluorescent markers, and challenging to detect their emission due to the strong extinction of the light within the sample. This creates a major challenge in detecting the fluorescent light of labeled cells in, for example, undiluted whole blood which has the characteristic crimson red color. One possible method to circumvent the problem of light extinction is to reduce the height of the microfluidic channel(s) that contain the dense sample. However the shallow depth of field and the relatively small field-of-view (FOV) of conventional optical microscopes result in an observation volume that is typically less than 1 μL. Mechanical scanning stages can increase the observed volume by capturing multiple images, either by moving the microscope objective or the sample itself; however these conventional microscopy based solutions would be rather costly, and would require capturing and digitally processing/stitching over 3,000 partially-overlapping images for screening a volume of e.g., ˜1 mL. Digitally processing this many partially-overlapping images is computationally intensive and could easily take many minutes or hours. One alternative method to image fluorescent micro-objects in optically dense media is to use spatially modulated excitation to increase the penetration of the light and use maximum intensity projection algorithms to boost the signal to noise ratio.
Other solutions focus on special sample preparation techniques and smart micro-fluidic chips that are able to extract the target cells with decent specificity and sensitivity from the medium before imaging them. All of these micro-fluidic approaches, however, rely on conventional fluorescent microscopes to image the entire active area of the chip and sometimes capture >5,000 images over a large FOV of 5-10 cm2 to detect the target cells of interest. To mitigate these challenges, there have been various efforts to increase the throughput of fluorescent imaging devices while also aiming to create compact, cost effective, and field-portable solutions for e.g., point-of-care applications. There remains a need for a cost effective fluorescent imaging platform that can rapidly detect fluorescent objects in bodily fluids that tend to extinguish light (e.g., whole blood).